Microspray column, mass spectrometer, and mass spectrometry

ABSTRACT

A problem is to provide a microspray column capable of increasing the ionization efficiency, and a high sensitive mass spectrometer and a mass spectrometric method using such a microspray column. For solving this problem, we provide a column for introducing a sample in an ionization source of a mass spectrometer (ESI/MS) being designed to ionize a sample molecule with electrospray and introduce it into an analyzer, has structural features of: (1) 0.5 μm or less in an inner diameter of a tip opening of the column; (2) 0.5 μm or more and 5 μm or less in a particle size of a column filler; and (3) fritless, a microspray column having the above (1) to (3), a mass spectrometer having such a microspray column in an ionization source, and a mass spectrometric method capable of performing a nonoflow electrospray with the microspray column.

TECHNICAL FIELD

The present invention relates to an improved technique in massspectrometry. In particular, the present invention relates to animproved technique of a mass spectrometer (ESI/MS) being designed toallow the introduction of a sample molecule with electrosprayionization, and a mass spectrometric method.

BACKGROUND ART

A mass spectrometer (hereinafter, referred to as “MS” (MassSpectrometer)) is roughly composed of: an “ionization source” forionizing a sample; an “analyzer” for separating ions according to theratio of mass/charge, represented by m/ze (wherein m: mass, z: chargenumber, and e: unit charge); and a “detection and recording part” ofions being separated.

The electrospray ionization technique generally referred to “ESI” (theabbreviation for Electrospray Ionization) is known as one of the methodsof ionizing and introducing a sample molecule to an analyzer of MS.

In this electrospray ionization technique, a spray is carried out byapplying the high voltage on a sample molecule being brought in ionicstate with acid or the like in a solvent.

This electrospray ionization technique is a technique for spraying asample molecule, which is brought into an ionic state by acid or thelike in the solution, by applying high voltage; forming liquid droplets(mist) in micron order, in which many solvent molecules are combinedwith multi-protonated molecules; and spraying nitrogen to dry and removethe solvent to ionize the sample molecule, followed by subjecting to theabove analyzer. As the charge number of ions being generated becomeslarge in this technique, it may be particularly useful in themeasurements of peptide and proteins, respectively.

Here, the electrospray ionization of the sample molecule in the aboveionization source of MS is performed by discharging and atomizing(spraying) the sample molecule in small quantities from a column formedof an elongated silica glass generally having an opening with a minuteaperture. This column will be referred to as a “microspray column”below.

FIG. 2 is a diagram that simplifies and expresses the configuration ofthe conventional electrospray ionization technique. The referencenumeral 10 denotes a conventional typical microspray column. On themicrospray column 10 being formed such that the tip portion thereof hasa cusp form, a large number of fillers 10 a such as chemical bond typesilica gels or the like having a particle size of about 50 μm is formed.In addition, the inner diameter d of the tip portion of the column isabout 10 to 15 μm. Furthermore, the outermost tip portion of themicrospray column 10 is loaded with a large-sized bead 10 b forpreventing the discharge of beads, which are also referred to as a flit.

This microspray column 10 is a constituent member of the ionizationsource of a mass spectrometer 11 and is arranged such that it extends tothe front of a pre-column 12 on which a high voltage is loaded. It isconfigured that fine droplets 14 containing the sample molecule areatomized from the tip portion of the microspray column 10 to theanalyzer 13 of the mass spectrometer 11.

However, in the conventional microspray column, the separationefficiency of a chromatograph was insufficient since the particlediameter of the filler in the column was large (generally about 50 μm).

In addition that the particle size of the filler was large, the innerdiameter of the tip opening of the above microspray column was alsolarge. Therefore, the discharge amount of the sample increased and theparticle size of charged liquid droplets formed by the spray was alsolarge. As a result, in the process in which the solvent was dried andvaporized, the efficiency of transferring charged electrons to thesample molecule in the solvent was not sufficient. In other words, theionization efficiency of the sample molecule was insufficient.

Furthermore, the microspray column was configured such that many areaswithout filling with the filler were formed near the tip opening,resulting in a large discharge amount of the sample and a large particlesize of the charged droplet formed with the spray.

Therefore, an object of the present invention is to provide a microspraycolumn capable of improving the ionization efficiency, and ahigh-sensitive mass spectrometer and a mass spectrometric method usingthis microspray column.

DISCLOSURE OF THE INVENTION

In order to solve the above-mentioned technical subject, the followingmeans are adopted in this invention.

At first, the microspray column for introducing a sample into anionization source of a mass spectrometer (ESI/MS) configured to performan electrospray ionization of the sample molecule and introduce thesample molecule into an analyzer was designed such that (1) the innerdiameter of a tip opening of the column is 0.5 μm or less, (2) aparticle size of the filler in the column is larger than 0.5 μm but notmore than 5 μm, and (3) there is no frit at all (fritless). Here, “frit”means the member of a major-diameter bead and other members for blockingthe bead loaded in the tip portion of the column. On the other hand,“fritless” means the configuration in which only the filler is filled inthe column without using the frit.

The particle size of the filler is a minimum diameter as much as itcannot be conventionally conceived. In addition, the inner diameter ofthe tip opening of the microspray column is small. Therefore, thedischarge amount of the sample can be substantially made small, comparedwith the conventional one. For this reason, the particle diameter of thecharged droplet formed by the spray can be miniaturized. As a result, itbecomes possible to increase the efficiency of transferring the chargedelectron in the solvent to the sample molecule. Therefore, an increasein ionization efficiency of the sample molecule becomes possible.

It is preferable to shape the tip portion of the microspray column intotapering form as much as possible. In addition, it is preferable tominimize the area of the tip portion of the microspray as much aspossible. This is because, when the area of the tip portion of thecolumn is too large, there is a phenomenon in which a large droplet isformed while the solution discharged from the column is being adhered onthe tip. Therefore, the liquid droplets can be prevented from becomingfine.

It is preferable that the inner diameter of the tip opening of thecolumn is 0.1 μm or more. This is because a high pressure is needed forthe discharge of a solution when the inner diameter of the tip openingis less than 0.1 μm.

In the present invention, furthermore, the present invention offers amass spectrometer characterized by comprising an ionization sourceconstructed such that a sample molecule contained in charged dropletsatomized from the above microspray column is ionized, and a massspectrometric method for performing an nanoflow electrospray using theabove microspray column. Here, the term “nanoflow electrospray” means atechnique capable of performing a stable electrospray ionization on asolution that contains a sample molecule to be fed at a flow rate in theorder of nano liters (nL/min) and introducing into an analyzer of a massspectrometer.

With this means, it is possible to provide a mass spectrometer havinghigh detection sensitivity and excellent ionization efficiency and amass spectrometric method.

As described above, the present invention is capable of improving theionization efficiency of a sample molecule and perfectly performing ananoflow electrospray by making fine charged liquid particles dischargedfrom the microspray column.

This technique has a technical meaning that the high sensitivitymeasurement of a high molecular weight compound such as peptide orprotein becomes possible because of an increase in the charge number ofthe ion to be generated.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an extended view around the tip portionof the microspray column of the present invention;

FIG. 2 is a diagram illustrating a simplified configuration of theconventional electrospray column technique;

FIG. 3(A) is a schematic diagram of the chromatograph of the massspectrometer when the conventional microspray column is used, and FIG.3(B) is a schematic diagram of the chromatograph of the massspectrometer when the microspray column of the present invention isused;

FIG. 4 is a chromatograph of the mass spectrometer obtained in theexample; and

FIG. 5 is a table of the whole amino acid sequence (609 amino acids)which is the digestive product of trypsin enzyme of human serum albumin.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferable embodiments of the present invention will be described withreference to the attached drawings.

FIG. 1 is an enlarged view of around the tip portion of the microspraycolumn of the present invention. In FIG. 1, the reference numeral 1denotes a microspray column (hereinafter, simply referred to as“column”) made of silica grass, which is shaped like an elongatedcylinder with a hollow formed therein.

This column 1 functions as a sample-installation column whichconstitutes an ionization source of a mass spectrometer (ESI/MS)designed to introduce a sample molecule into the analyzer after ionizingthe sample compound with electrospray. As shown in FIG. 1, the tipportion 1 a of the column 1 has a cusp form which tapers off gradually,and a tip opening 1 b having a specific diameter for discharging asample solution and atomizing the sample solution toward an analyzer ofa mass spectrometer not shown in the figure is formed in the outermosttip portion.

Furthermore, in the inside of the column 1, a filler 2 functioned as asorbent at the time of separating a sample is filled up with uniformdensity. In the tip opening 1 b, it is configured such that a frit isnot loaded. That is, the column 1 of the present invention is a fritlesscolumn.

Here, in the present invention, the inner diameter D₁ of the tip opening1 b of the column 1 is 0.5 μm or less, preferably 0.1 μm or more and 0.5μm or less. The particle size D₂ of the above filler 2 is more than 0.5μm and 5 μm or less.

The reason of setting the inner diameter D₁ of the tip opening 1 b to0.5 μm or less (D₁≦0.5 μm) is that a charged droplet containing a samplemolecule separated from the column 1 and atomized from the tip opening 1b is sufficiently made smaller to increase the ionization efficiency ofthe sample molecule with certainty.

Specifically, the inner aperture D₁ of tip opening 1 b is set to 0.5 μmor less to allow the mass spectrometer to generate a large charge numberof ions in the sample molecule enough to realize the high-sensitivemeasurement of a high molecular weight compound such as peptide orprotein using a mass spectrometer.

In addition, the reason of defining the particle size D₂ of the filler 2to more than 0.5 μm and 5 μm or less (0.5 μm<D₂≦5 μm) is that forpreventing the filer 2 from being discharged through the tip opening 1 bof 0.5 μm or less in inner diameter. The particle size D₂ of the filleris made larger than the inner diameter D₁ of the above tip opening 1(D₁<D₂), while the particle size D₂ of the filler 2 is made smaller than5 μm or less to increase the total surface area of the whole filler 2.Furthermore, as the particle size D₂ of the filler 2 is defined as 5 μmor less, the results can be obtained within a short time with a smallamount of an eluent at a nanoflow level. In addition, a chemical bondtype silica gel (e.g., C18 having a large absorbency) may be used as thefiller 2.

Here, FIG. 3 is a schematic diagram for making a comparison between thechromatograph (A) of the mass spectrometer at the time of using theconventional microspray column and the chromatograph (B) of the massspectrometer at the time of using the microspray column of the presentinvention.

As shown in FIG. 3, the microspray column of the present invention showsa sharp peak in the chromatograph of the mass spectrometer. Moreconcretely, reading of the rising peak and also no tailing in the secondhalf of a peak cannot be , and, more specifically, the tendency ofgenerating a sharp peak that exceeds a detection limit can be notablyappeared.

The sensitivity of the mass spectrometer is concentration-dependent, sothat the signal strength increases as the peak height increases.

Therefore, using the microspray column of this invention, thesensibility and the resolution of a mass spectrometer become high. Inaddition, it becomes possible to raise the rate of decoding an aminoacid sequence (sequence coverage), exponentially.

EXAMPLE

A solution including a digestive product of a trypsin enzyme derivedfrom human serum albumin (a molecular weight of 65,000 to 70,000) at aconcentration of 50 femto mole or less was atomized from a microspraycolumn of the present invention under the conditions in which the tipdiameter of the column was 0.5 μm or less, fritless, and the particlesize of the filler was 1.0 μm.

The chromatograph of the mass spectrometer obtained in the presentexample is shown in FIG. 4. As shown in the schematic diagram of FIG. 3,the sharp peak without reading or tailing was also obtained by theactual experimental findings as shown in the schematic diagram of FIG.3, so that the decipherment of an amino acid sequence could be raisedexponentially.

FIG. 5 shows a total amino acid sequence table (609 amino acids) of theabove digestive product. In this example, the 573 amino acid sequencesexcept of 36 amino-acid portion surrounded by a square enclosure in FIG.5 were deciphered. The rate of a decipherment was dramatically as highas 94% (573/609).

INDUSTRIAL APPLICABILITY

According to the microspray column of the present invention, theparticle size of the filler is a minimum diameter in addition to makethe inner diameter of the microspray column equal to a predetermineddiameter or less. Therefore, the discharge amount of a sample solutionper unit time can be sharply lessened as compared with the conventionalone, so that the particle size of charged droplets formed by sprayingcan be made finer. As a result, the efficiency of transferring theelectrons charged in the solvent to the sample molecule can beextensively improved and the ionization efficiency of the samplemolecule can be increased. Furthermore, in the present invention, it ischaracterized by comprising an ionization source designed to ionize thesample molecule to be contained in the charged droplets atomized fromthe above microspray, so that a mass spectrometer having excellentionization efficiency and high detection sensitivity can be provided.Furthermore, according to the present invention, a mass spectrometricmethod that surely performs a nanoflow electrospray and nano-LC gradientanalysis by the above microspray column, so that a high sensitivemeasurement of high molecular weight compound such as peptide andprotein can be performed, positively. Consequently, an extensiveimprovement in the rate of an amino acid decipherment of peptide orprotein can be attained.

1. A column for introducing a sample in an ionization source of a massspectrometer (ESI/MS) being designed to ionize a sample molecule withelectrospray and introduce it into an analyzer, comprising structuralfeatures of: (1) 0.5 μm or less in an inner diameter of a tip opening ofthe column; (2) 0.5 μm or more and 5 μm or less in a particle size of acolumn filler; and (3) fritless.
 2. A mass spectrometer, comprising: anionization source which is constructed such that a sample molecule inliquid droplets atomized from the microspray column described in claim 1is ionized.
 3. A mass spectrometric method, wherein a nonoflowelectrospray is performed using the microspray column described in claim1.